1. Field of the Invention
The present invention relates generally to the fields of enzymology and steroid chemistry. More specifically, the present invention relates to an enzymatic method of producing 7-dehydropregnenolone, vitamin D3-like compounds and derivatives thereof.
2. Description of the Related Art
The skin, the largest body organ, maintains internal homeostasis by not only separating the external environment from the internal milieu, but, also, through its immune and neuroendocrine activities (1-3). Cutaneous elements can, in addition, have powerful systemic actions, as is the case for vitamins D3 (1), which regulate calcium metabolism and modulate immune and neuroendocrine activities and proliferation and differentiation in cells of different lineage (4-6). Vitamin D3 is formed from the precursor steroid 7-dehydrocholesterol (7-DHC) localized mostly on the plasma membrane of basal epidermal keratinocytes which has 80% of skin 7-dehydrocholesterol content. Upon stimulation with photons of ultraviolet light B, i.e, light having a wavelength of 290-320 nm, 7-dehydrocholesterol undergoes photolysis to generate previtamin D3, which at normal skin temperature undergoes internal rearrangement to convert into vitamin D3 (4,7).
Cytochrome P450scc is a product of the CYP11A1 locus, thought until recently to use solely cholesterol as substrate, to hydroxylate and cleave the side chain at a single active site on the cytochrome to produce pregnenolone (8). Electrons for the hydroxylations are provided by NADPH through the electron transfer proteins adrenodoxin reductase and adrenodoxin (8-9) and cholesterol via transport StAR protein or MLN64 (20-21). The cholesterol substrate for P450scc is transported into mitochondria by specific cholesterol transporting proteins, StAR in testis, adrenal and ovary, and likely MLN64 in the placenta (20-21,23). Cholesterol transport by MLN64 in mitochondria may require proteolytic processing to release the 27-kDa cholesterol-binding domain from the full-length form associated with late endosomes (21,23). There also is evidence that the full-length 55-kDa form of MLN64 is associated with placental mitochondria (24). This biochemical pathway may be operative in the skin, since it expresses the related CYP11A1, CYP17, CYP21A2 and MC2-R genes (10).
Furthermore, skin and skin cells can rapidly and selectively metabolize progesterone and deoxycorticosterone (DOC) to a number of intermediates that include deoxycorticosterone, 18-hydroxydeoxycorticosterone and corticosterone, consistent with active local steroidogenesis (11-14). Recent information indicates an active P450scc system present in immortalized sebocytes. Cultured sebocytes converted 22R-hydroxycholesterol to 17-hydroxypregnenolone (22). Additionally, P450scc was detected by immunocytochemistry in human epidermis and hair follicle (22).
Interest in the P450scc system has been renewed by recent findings in patients with the rare Smith-Lemli-Opitz syndrome (SLOS) whose cholesterol synthesis from 7-dehydrocholesterol is impaired from deficiency of the 7-DHC-delta-7 reductase (15,16). Patients with Smith-Lemli-Opitz syndrome have noticeable amounts of 7-dehydropregnenolone (7-DHP) and its metabolites suggesting enzymatic production from 7-dehydrocholesterol (17,18). Furthermore, in most recent studies with an in vitro system of reconstituted P450scc, 7-dehydrocholesterol and vitamin D3 were found to serve as alternate substrates for cytochrome P450scc (19).
Epidermal availability of 7-dehydrocholesterol in conjunction with the presence of an active P450scc system makes it likely that 7-dehydropregnenolone is produced in the skin, albeit at low basal rates. Since the unsaturated B ring of 7-dehydropregnenolone is prone to breakage of the 9,10 carbon bond by UVB, and to further temperature dependent conversion, 7-dehydropregnenolone is probably transformed into the vitamin D3-like compound 5Z,7E-3β-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one or vitDL (27). This conversion may be the explanation for the lack of elevation of vitamin D3 levels in spite of 7-dehydrocholesterol tissue accumulation in Smith-Lemli-Opitz syndrome patients (30). In fact, the skin, exposed to solar radiation, would be the site of choice for production of vitDL. Interestingly, native vitDL and its derivatives are known to possess immunomodulatory and anti-tumor properties (27).
The inventors have recognized that skin presents the unique situation of having readily available all the potential substrates for P450scc, e.g., cholesterol, 7-dehydrocholesterol and vitamins D2 and D3. This provides the background for the systematic investigation of cutaneous expression for each of the components of the P450scc enzymatic system. Furthermore, this also provides a basis to develop and test in vitro enzyme systems that use these substrates to produce new and useful steroids.
The prior art is deficient in the lack of an efficient and potentially large-scale enzymatic production of 7-dehydropregnenolone, vitamin D2-like- and vitamin D3-like compounds and derivatives thereof. Specifically, the prior art is deficient in the lack of a cytochrome P450scc enzyme system using 7-dehydrocholesterol, vitamin D3 or vitamin D2 as substrates to produce these compounds. The present invention fulfills this long-standing need and desire in the art.